Certain key cellular constituents, which we term "common proteins", are highly conserved across the major divisions of life and belong to vast protein classes that have functionally diverged into many hierarchically arranged subgroups. Thus each of these proteins manifests variations on recurring structural and/or mechanistic themes. These include, for example, AAA+ ATPases, P-loop GTPases and protein kinases, which were the focus of our preliminary studies due to their biomedical significance. AAA+ ATPases are associated with hereditary spastic paraplegia and the neurologic disorders torsin dystonia, Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. The GTPase Ras plays a key role in cancer, and protein kinases are important cancer and diabetes drug targets. A major goal of structural biology is to understand protein mechanisms in atomic-detail within the context of the living cell. Despite remarkable progress in determining protein structures, however, many aspects of underlying protein mechanisms remain unclear. Determining these mechanisms is a daunting task that will require many carefully chosen hypotheses and experiments to sort out. Such hypotheses are not formulated in a conceptual vacuum, however, but rather are based on clues obtained from preliminary observations. The functional constraints imposed on proteins during evolution are a potential source of information in this regard, inasmuch as these are due to and thus reflect underlying mechanisms. Moreover, because natural selection imposes these constraints on the genomic sequences of living organisms within their native environments, such information lacks the artifactual biases sometimes associated with in vitro experimental systems or with in vivo cell cultures and may reveal functionally critical features that have been overlooked due to the inherent limitations of current experimental methods. Thus the broad, long-range goal of this project is to characterize the functional constraints imposed on common proteins and to thereby provide clues to their underlying mechanisms as an aid to experimental design. Over the past decade we have developed and applied statistically rigorous procedures for characterizing complex patterns of sequence conservation across and within subgroups of related proteins. Using these and other approaches this project will accomplish the following specific aims: (i) Detect and very accurately align as many sequences as possible from several common protein classes, (ii) Identify, categorize and quantify the functional constraints acting on these proteins through statistical analysis of the alignments, (iii) Identify structural and chemical features associated with these constraints, (iv) Similarly analyze proteins that functionally interact with these proteins. And (v) propose molecular mechanisms based on these analyses.